The success of neuronal cell replacement therapy depends on the ability of transplanted cells to synaptically integrate with host tissue. Complete integration requires that neurons can both send and receive synaptic information, as well as to modify their synaptic strength in response to changes in the cellular behavior of synaptically connected neurons. Due to limited cell tracking and stimulation techniques, previous reports have shown only that transplanted neurons can receive information from host neurons via synaptic stimulation. Thus, no direct evidence exists for their ability to send information to host cells or undergo synaptic plasticity. To test these hypotheses, we propose to use the light-activated Channelrhodopsin-2 (ChR2) ion channel linked to the mCherry fluorophore in human embryonic stem cell (hESC)-derived neurons. Following transplantation of hESC-derived forebrain-patterned neurons to the mouse hippocampus, we will use light stimulation to selectively activate human neurons while recording mouse cells. We hypothesize that light stimulation (and subsequent action potential generation) will give rise to robust synaptic activation of host neurons. Application of various light stimulus protocols will then test whether human neurons can trigger short-term and long-term forms of synaptic plasticity in host cells. In parallel, we will perform similar experiments on mixed cultures of ChR2-expressing and non-expressing hESC-derived neurons. Here, application of light stimulation protocols will test whether hESC-derived neurons can undergo post-synaptic changes required for enduring changes in synaptic efficacy. Together, these data seek to provide evidence of the ability of human neurons to act as a fully functional unit within a neural network in host tissues. PUBLIC HEALTH RELEVANCE: Successful neuronal cell replacement is thought to rely on the integration of transplanted cells with host tissue via the formation of synaptic connections. Until recently, technical limitations have prevented the determination of whether stem cell-derived neurons can send information to host cells or undergo changes in synaptic efficacy that are necessary for true circuit integration. The proposed research will use the newly characterized light activated Channelrhodopsin-2 in human embryonic cell-derived neurons to regulate the excitability of transplanted neurons to test these hypotheses.